Current Topic pubs.acs.org/biochemistry
Thymidine Kinase 2 Enzyme Kinetics Elucidate the Mechanism of Thymidine-Induced Mitochondrial DNA Depletion Ren Sun and Liya Wang* Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, SE-750 07 Uppsala, Sweden ABSTRACT: Mitochondrial thymidine kinase 2 (TK2) is a nuclear geneencoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis− Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in lateonset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.
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logues.3−5 TK2 is present in all cells that contain mitochondria, and the levels of TK2 correlated to the mitochondrial content of the cells or tissues.2 A recent study showed that TK2 is upregulated during the stationary phase growth of cultured cells.6 Deficiency in TK2 activity due to genetic mutations causes devastating mitochondrial diseases, which are characterized as tissue-specific mtDNA depletion and/or deletion. Mitochondrial DNA depletion syndrome (MDS) is characterized by a severe and tissue-specific reduction in the mtDNA copy number in the absence of qualitative defects in mtDNA.7 MDS caused by TK2 mutations affected mainly liver and skeletal muscle, but in some cases, multiple tissues were involved.8−10 Mutations in the TK2 gene are also the genetic causes of late-onset autosomal recessive progressive external ophthalmoplegia (arPEO).11 The subject of this review is the substrate specificity, enzyme kinetics, and regulation of the TK2 enzyme, as well as the mechanism of thymidine overloadinduced mtDNA depletion.
itochondria are dynamic organelles that undergo constant changes, e.g., fission and fusion, during the lifetime of the cell. Mitochondria contain multiple copies of circular mitochondrial DNA (mtDNA) molecules, which encode 13 essential subunits of the mitochondrial respiratory chain complexes. To maintain a sustainable mtDNA copy number, a constant supply of precursors, i.e., dNTPs, is essential because mtDNA replicates in a manner that is independent of the cell cycle. A limitation of one or more dNTPs will stall mtDNA synthesis and result in mtDNA depletion, which leads to mitochondrial diseases.1 In dividing cells and/or tissues, mitochondrial dNTPs are synthesized via the cytosolic de novo pathway and the salvage pathway; however, in nondividing cells, the dNTPs required for mtDNA replication are mainly generated in organello via thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK). TK2 and dGK catalyze the phosphorylation of all four natural deoxynucleosides to their respective monophosphates using ATP or other nucleoside triphosphates as phosphate donors. Both enzymes are essential for mtDNA precursor synthesis and mitochondrial function in many cell and tissue types.2 Thymidine kinase 2 (TK2, EC 27.1.21) catalyzes the transfer of the γ-phosphate group from ATP to the 5′-hydroxyl group of thymidine (dT), deoxycytidine (dC), or deoxyuridine (dU) to form their corresponding monophosphates. TK2 also phosphorylates a number of pyrimidine nucleoside analogues, such as zidovudine (AZT) used in anti-HIV therapy, and thus may play an important role in the mitochondrial toxicity observed in antiviral and anticancer therapies using nucleoside ana© 2014 American Chemical Society
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TK2 ENZYME KINETICS The first extensive kinetic analysis was performed using the purified enzyme obtained from human leukemic spleen.12 It was shown that with dC and dU as substrates, the reactions Received: June 4, 2014 Revised: September 11, 2014 Published: September 12, 2014 6142
dx.doi.org/10.1021/bi5006877 | Biochemistry 2014, 53, 6142−6150
Biochemistry
Current Topic
followed hyperbolic Michaelis−Menten kinetics, with Km (micromolar) and Vmax/Km (units per milligram per micromolar) values of 36 and 25 for dC and 6 and 115 for dU, respectively. However, the phosphorylation of dT exhibited negative cooperativity with Hill coefficients in the range of 0.3− 0.5, indicating that the affinity of dT for TK2 decreases with an increase in concentration as shown by the biphasic Hofstee plot. The Km value was 0.3 μM when the dT concentration was 8 μM) than at low dT concentrations (Figure 1). The specific activity
TK2 belongs to the enzyme family that includes mitochondrial deoxyguanosine kinase (dGK), cytosolic deoxycytidine kinase (dCK), and Drosophila melanogaster deoxynucleoside kinase (Dm-dNK). The three-dimensional (3D) structures of dGK, dCK, and Dm-dNK have been determined but not the TK2 structure. Substrate and inhibitor binding sites have been studied in detail with dGK, dCK, and Dm-dNK.19−22 However, there is a fundamental lack of structural information about TK2; hence, the number of substrate binding sites per active enzyme is not known, which is a real barrier to achieving a clear understanding of its biological function.
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REGULATION OF TK2 ACTIVITY BY SUBSTRATES, SUBSTRATE ANALOGUES, AND FEEDBACK INHIBITORS Substrate dT and dC phosphorylations are regulated by a different kinetic mechanisms, and thus, the enzyme has different catalytic efficiencies for dT and dC, as described above. The negative cooperative kinetics with dT suggest that under physiological conditions, e.g., low dT concentrations (
Thymidine Kinase 2 Enzyme Kinetics Elucidate the Mechanism of Thymidine-Induced Mitochondrial DNA Depletion Ren Sun and Liya Wang* Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, SE-750 07 Uppsala, Sweden ABSTRACT: Mitochondrial thymidine kinase 2 (TK2) is a nuclear geneencoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis− Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in lateonset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.
M
logues.3−5 TK2 is present in all cells that contain mitochondria, and the levels of TK2 correlated to the mitochondrial content of the cells or tissues.2 A recent study showed that TK2 is upregulated during the stationary phase growth of cultured cells.6 Deficiency in TK2 activity due to genetic mutations causes devastating mitochondrial diseases, which are characterized as tissue-specific mtDNA depletion and/or deletion. Mitochondrial DNA depletion syndrome (MDS) is characterized by a severe and tissue-specific reduction in the mtDNA copy number in the absence of qualitative defects in mtDNA.7 MDS caused by TK2 mutations affected mainly liver and skeletal muscle, but in some cases, multiple tissues were involved.8−10 Mutations in the TK2 gene are also the genetic causes of late-onset autosomal recessive progressive external ophthalmoplegia (arPEO).11 The subject of this review is the substrate specificity, enzyme kinetics, and regulation of the TK2 enzyme, as well as the mechanism of thymidine overloadinduced mtDNA depletion.
itochondria are dynamic organelles that undergo constant changes, e.g., fission and fusion, during the lifetime of the cell. Mitochondria contain multiple copies of circular mitochondrial DNA (mtDNA) molecules, which encode 13 essential subunits of the mitochondrial respiratory chain complexes. To maintain a sustainable mtDNA copy number, a constant supply of precursors, i.e., dNTPs, is essential because mtDNA replicates in a manner that is independent of the cell cycle. A limitation of one or more dNTPs will stall mtDNA synthesis and result in mtDNA depletion, which leads to mitochondrial diseases.1 In dividing cells and/or tissues, mitochondrial dNTPs are synthesized via the cytosolic de novo pathway and the salvage pathway; however, in nondividing cells, the dNTPs required for mtDNA replication are mainly generated in organello via thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK). TK2 and dGK catalyze the phosphorylation of all four natural deoxynucleosides to their respective monophosphates using ATP or other nucleoside triphosphates as phosphate donors. Both enzymes are essential for mtDNA precursor synthesis and mitochondrial function in many cell and tissue types.2 Thymidine kinase 2 (TK2, EC 27.1.21) catalyzes the transfer of the γ-phosphate group from ATP to the 5′-hydroxyl group of thymidine (dT), deoxycytidine (dC), or deoxyuridine (dU) to form their corresponding monophosphates. TK2 also phosphorylates a number of pyrimidine nucleoside analogues, such as zidovudine (AZT) used in anti-HIV therapy, and thus may play an important role in the mitochondrial toxicity observed in antiviral and anticancer therapies using nucleoside ana© 2014 American Chemical Society
■
TK2 ENZYME KINETICS The first extensive kinetic analysis was performed using the purified enzyme obtained from human leukemic spleen.12 It was shown that with dC and dU as substrates, the reactions Received: June 4, 2014 Revised: September 11, 2014 Published: September 12, 2014 6142
dx.doi.org/10.1021/bi5006877 | Biochemistry 2014, 53, 6142−6150
Biochemistry
Current Topic
followed hyperbolic Michaelis−Menten kinetics, with Km (micromolar) and Vmax/Km (units per milligram per micromolar) values of 36 and 25 for dC and 6 and 115 for dU, respectively. However, the phosphorylation of dT exhibited negative cooperativity with Hill coefficients in the range of 0.3− 0.5, indicating that the affinity of dT for TK2 decreases with an increase in concentration as shown by the biphasic Hofstee plot. The Km value was 0.3 μM when the dT concentration was 8 μM) than at low dT concentrations (Figure 1). The specific activity
TK2 belongs to the enzyme family that includes mitochondrial deoxyguanosine kinase (dGK), cytosolic deoxycytidine kinase (dCK), and Drosophila melanogaster deoxynucleoside kinase (Dm-dNK). The three-dimensional (3D) structures of dGK, dCK, and Dm-dNK have been determined but not the TK2 structure. Substrate and inhibitor binding sites have been studied in detail with dGK, dCK, and Dm-dNK.19−22 However, there is a fundamental lack of structural information about TK2; hence, the number of substrate binding sites per active enzyme is not known, which is a real barrier to achieving a clear understanding of its biological function.
■
REGULATION OF TK2 ACTIVITY BY SUBSTRATES, SUBSTRATE ANALOGUES, AND FEEDBACK INHIBITORS Substrate dT and dC phosphorylations are regulated by a different kinetic mechanisms, and thus, the enzyme has different catalytic efficiencies for dT and dC, as described above. The negative cooperative kinetics with dT suggest that under physiological conditions, e.g., low dT concentrations (
